Neutralization of Omicron BA.1, BA.2, and BA.3 SARS-CoV-2 by 3 doses of BNT162b2 vaccine

The newly emerged Omicron SARS-CoV-2 has several distinct sublineages including BA.1, BA.2, and BA.3. BA.1 accounts for the initial surge and is being replaced by BA.2, whereas BA.3 is at a low prevalence at this time. Here we report the neutralization of BNT162b2-vaccinated sera (collected 1 month after dose 3) against the three Omicron sublineages. To facilitate the neutralization testing, we have engineered the complete BA.1, BA.2, or BA.3 spike into an mNeonGreen USA-WA1/2020 SRAS-CoV-2. All BNT162b2-vaccinated sera neutralize USA-WA1/2020, BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s with titers of >20; the neutralization geometric mean titers (GMTs) against the four viruses are 1211, 336, 300, and 190, respectively. Thus, the BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s are 3.6-, 4.0-, and 6.4-fold less efficiently neutralized than the USA-WA1/2020, respectively. Our data have implications in vaccine strategy and understanding the biology of Omicron sublineages.

T he severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has recently emerged as the fifth variant of concern (VOC) after the previous Alpha, Beta, Gamma, and Delta VOCs. As of 12 March 2022, The Omicron variant includes 3 sublineages: BA.1, BA.2, and BA. 3. After initial identification in South Africa in November 2021, the Omicron sublineage BA.1, and its derivative BA.1.1 (containing an extra spike R346K substitution), became dominant worldwide. Subsequently, sublineage BA.2 sharply increased its prevalence in many countries, including Demark, the Philippines, South Africa, and Belgium. In the United States, the prevalence of BA.2 increased from 0.4% on January 22 to 23.1% on March 12, 2022 (https://covid.cdc.gov/covid-data-tracker/#variant-proportions). Studies have also shown that BA.2 may be approximately 30% more transmissible than BA.1 1 , but it does not appear to cause more severe disease 2 . Compared with the BA.1 and BA.2 sublineages, the prevalence of BA.3 has remained low, with a total of 613 BA.3 sequences in the GISAID database (https:// www.gisaid.org/) as of 12 March 2022. Recent studies showed that BA.1 evades vaccine-and non-Omicron infection-elicited neutralization 3,4 , which, together with increased transmissibility, may partially account for the variant replacement from the previous Delta to the current Omicron.
BNT162b2 is an mRNA vaccine that encodes a stabilized prefusion full-length spike glycoprotein from the original SARS-CoV-2 Wuhan-Hu-1 isolate. BNT162b2 mRNA has been approved for vaccination of people ≥16-year-old and has also been authorized under emergency use provision for immunization of children 5-to 15-year-old by the US Food and Drug Administration. Since BA.1, BA2, and BA.3 have distinct sets of mutations in their spike glycoproteins (Fig. S1a), laboratory results are urgently needed to evaluate the susceptibility of the three Omicron sublineages to vaccine-elicited neutralization. In this work, we show the neutralization of BNT162b2-vaccinated sera (collected 1 month after dose 3) against the three Omicron sublineages.

Results and discussions
To determine the susceptibility of Omicron sublineages to BNT162b2-elicited neutralization, we engineered the complete BA.1 (GISAID EPI_ISL_6640916), BA.2 (GISAID EPI_ISL_6795834.2), or BA.3 (GISAID EPI_ISL_7605591) spike into the mNeonGreen (mNG) reporter USA-WA1/2020, an SARS-CoV-2 strain isolated in January 2020 (Fig. S1a). The mNG gene was inserted into the openreading-frame-7 (ORF7) of the viral genome to enable the development of a fluorescent focus reduction neutralization test (FFRNT) 4 . Our historical data set indicate that the high-throughput FFRNT produces comparable neutralization results as the gold standard plaque-reduction neutralization test (PRNT; using SARS-CoV-2 without the mNG reporter) when analyzing BNT162b2vaccinated human sera (Fig. S2). Thus, FFRNT can reliably be used to measure antibody neutralization.
Using a panel of human sera collected at 1-month post dose 3 (PD3) of BNT162b2 vaccine 3 (Fig. S3), we determined the 50% fluorescent focus-reduction neutralization titers (FFRNT 50 ) against recombinant USA-WA1/2020 and Omicron sublineage-spike mNG SARS-CoV-2s. We chose the PD3 sera, rather than post dose 2 sera, because (i) two doses of BNT162b2 did not elicit robust neutralization against Omicron BA.1 3 and (ii) many individuals have already received 3 doses of BNT162b2. The PD3 sera neutralized USA-WA1/2020, BA.1-, BA.2-, and BA.3-spike mNG viruses with geometric mean titers (GMTs) of 1211, 336, 300, and 190, respectively ( Fig. 1). Although all PD3 sera neutralized recombinant viruses with titers of >20, the neutralizing GMTs against BA.1-, BA.2-, and BA.3-spike mNG viruses were 3.6-, 4.0-, and 6.4-fold lower than the GMT against the mNG USA-WA1/2020, respectively (Fig. 1). The results support two conclusions. First, BA.1 and BA.2 spikes evade the neutralization of PD3 sera to comparable degrees. Similar reduced neutralization of BA.1 and BA.2 sublineages was reported 5,6 ; however, the reduction folds varied among studies, likely due to different serum specimens and neutralization assay protocols. These results may also imply that the observed increasing prevalence of BA.2 over BA.1 among circulating strains was not driven by the difference in antibody neutralization after vaccination, but by other factors, such as differences in viral replication 7 and transmission 8 , or other potential immune evasion mechanisms.  Despite the reduced neutralization of Omicron sublineages, other immune effectors, such as T cells and non-neutralizing antibodies that mediate antibody-dependent cytotoxicity, also contribute to the protection against severe COVID-19. The majority of T cell epitopes after vaccination or natural infection are preserved against Omicron spikes 9 . Indeed, 3 doses of BNT162b2 have conferred efficacy against Omicron disease; however, recent real-world effectiveness data showed a waning of protection against symptomatic infection caused by Omicron. The waning of protection was more modest against severe disease caused by Omicron, with overall efficacy remaining high 3-6 months after dose 3 [10][11][12][13][14] . Studies are underway to determine the extended durability of the PD3 neutralization against Omicron sublineages and future variants. These laboratory investigations, together with real-world vaccine effectiveness data, will continue to guide 4th dose vaccine strategy for optimal breadth and duration of protection.

Methods
Ethical statement. All virus work was performed in a biosafety level 3 (BSL-3) laboratory with redundant fans in the biosafety cabinets at The University of Texas Medical Branch at Galveston. All personnel wore powered air-purifying respirators (Breathe Easy, 3M) with Tyvek suits, aprons, booties, and double gloves.
Cells. Vero E6 (ATCC® CRL-1586) was purchased from the American Type Culture Collection (ATCC, Bethesda, MD), and maintained in a high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, South Logan, UT) and 1% penicillin/ streptomycin at 37°C with 5% CO 2 . All culture media and antibiotics were purchased from ThermoFisher Scientific (Waltham, MA). The cell line was tested negative for mycoplasma.
Human serum. Serum samples were collected from BNT162b2 vaccinees participating in the phase 1 portion of the ongoing phase 1/2/3 clinical trial (Clinical-Trials.gov identifier: NCT04368728). The protocol and informed consent were approved by institutional review boards for each of the investigational centers participating in the study. The study was conducted in compliance with all International Council for Harmonisation Good Clinical Practice guidelines and the ethical principles of the Declaration of Helsinki. The primary outcomes for phase 1 were reported previously 15,16 . BNT162b2-vaccinated serum (n = 22) was collected on the day of the third dose of BNT162b2, which was administered at 7.9-8.8 months after the second dose; and sera collected 1 month after the third dose were used in the present study for neutralization testing. Table S1 summarizes the patient information (e.g., age and gender) and sample collection time points. The information for the serum panels were reported previously 17,18 .
Construction and characterization of recombinant Omicron sublineage spike mNG SARS-CoV-2s. Recombinant Omicron BA.1-, BA.2-, and BA.3-spike mNG SARS-CoV-2s were constructed by engineering the complete spike gene from Omicron sublineages into an infectious cDNA clone of mNG USA-WA1/2020 19 (Fig. S1a). All spike mutations, deletions, and insertions were introduced into the infectious cDNA clone of mNG USA-WA1/2020 using PCR-based mutagenesis as previously described 20 . The BA.1, BA.2, and BA.3 spike sequences were based on GISAID EPI_ISL_6640916, EPI_ISL_6795834.2, and EPI_ISL_7605591, respectively. The full-length cDNA of viral genome containing the complete Omicron spike was assembled via in vitro ligation. The resulting full-length cDNA was used as a template for in vitro transcription of genome-length viral RNA. The in vitro transcribed viral RNA was electroporated into Vero E6 cells. On day 3 post electroporation, the original viral stock (P0) was harvested from the electroporated cells. The P0 virus was amplified for another round on Vero E6 cells to produce the P1 stock for neutralization testing. The infectious titer of the P1 virus was quantified by fluorescent focus assay on Vero E6 cells (Fig. S1b). We sequenced the complete spike gene of the P1 virus to ensure no undesired mutations. Only the P1 viruses were used for the neutralization test. The protocols for the mutagenesis of mNG SARS-CoV-2 and virus production were reported previously 21 . To determine the specific infectivity, we quantified the P1 virus stocks for their fluorescent focus units (FFU) and genomic RNA contents by fluorescent focus assay on Vero E6 cells and RT-qPCR, respectively. The methods for fluorescent focus assay and RT-qPCR were reported previously 4,22 . The specific infectivity of each virus was measured by the genomic RNA-to-FFU ratios (genome/FFU).
Fluorescent focus reduction neutralization test. Neutralization titers of human sera were measured by FFRNT using the USA-WA1/2020, BA.1-, BA.2-, and BA.3spike mNG SARS-CoV-2s. The details of the FFRNT protocol were reported previously 4 . Briefly, 2.5 × 10 4 Vero E6 cells per well were seeded in 96-well plates (Greiner Bio-one™). The cells were incubated overnight. On the next day, each serum was 2-fold serially diluted in the culture medium with the first dilution of 1:20 (final dilution range of 1:20 to 1:20,480). The diluted serum was incubated with 100-150 FFUs of mNG SARS-CoV-2 at 37°C for 1 h, after which the serumvirus mixtures were loaded onto the pre-seeded Vero E6 cell monolayer in 96-well plates. After 1 h infection, the inoculum was removed and 100 μl of overlay medium (supplemented with 0.8% methylcellulose) was added to each well. After incubating the plates at 37°C for 16 h, raw images of mNG foci were acquired using Cytation TM 7 (BioTek) armed with 2.5× FL Zeiss objective with a wide-field of view and processed using the Gene 5 software settings (GFP [469,525] threshold 4000, object selection size 50-1000 µm). The foci in each well were counted and normalized to the non-serum-treated controls to calculate the relative infectivities. The FFRNT 50 value was defined as the minimal serum dilution that suppressed >50% of fluorescent foci. The neutralization titer of each serum was determined in duplicate assays, and the geometric mean was taken. All attempts at replication were successful. Table S1 summarizes the FFRNT 50 results. Data were initially plotted in GraphPad Prism 9 software and assembled in Adobe Illustrator.
Statistics. The nonparametric Wilcoxon matched-pairs signed-rank test was used to analyze the statistical significance in Fig. 1.